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Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS
Next-generation sequencing (NGS) has raised a growing interest in phage display research. Sequencing depth is a pivotal parameter for using NGS. In the current study, we made a side-by-side comparison of two NGS platforms with different sequencing depths, denoted as lower-throughput (LTP) and higher...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10049078/ https://www.ncbi.nlm.nih.gov/pubmed/36982469 http://dx.doi.org/10.3390/ijms24065396 |
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author | Sloth, Ane Beth Bakhshinejad, Babak Stavnsbjerg, Camilla Rossing, Maria Kjaer, Andreas |
author_facet | Sloth, Ane Beth Bakhshinejad, Babak Stavnsbjerg, Camilla Rossing, Maria Kjaer, Andreas |
author_sort | Sloth, Ane Beth |
collection | PubMed |
description | Next-generation sequencing (NGS) has raised a growing interest in phage display research. Sequencing depth is a pivotal parameter for using NGS. In the current study, we made a side-by-side comparison of two NGS platforms with different sequencing depths, denoted as lower-throughput (LTP) and higher-throughput (HTP). The capacity of these platforms for characterization of the composition, quality, and diversity of the unselected Ph.D.(TM)-12 Phage Display Peptide Library was investigated. Our results indicated that HTP sequencing detects a considerably higher number of unique sequences compared to the LTP platform, thus covering a broader diversity of the library. We found a larger percentage of singletons, a smaller percentage of repeated sequences, and a greater percentage of distinct sequences in the LTP datasets. These parameters suggest a higher library quality, resulting in potentially misleading information when using LTP sequencing for such assessment. Our observations showed that HTP reveals a broader distribution of peptide frequencies, thus revealing increased heterogeneity of the library by the HTP approach and offering a comparatively higher capacity for distinguishing peptides from each other. Our analyses suggested that LTP and HTP datasets show discrepancies in their peptide composition and position-specific distribution of amino acids within the library. Taken together, these findings lead us to the conclusion that a higher sequencing depth can yield more in-depth insights into the composition of the library and provide a more complete picture of the quality and diversity of phage display peptide libraries. |
format | Online Article Text |
id | pubmed-10049078 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-100490782023-03-29 Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS Sloth, Ane Beth Bakhshinejad, Babak Stavnsbjerg, Camilla Rossing, Maria Kjaer, Andreas Int J Mol Sci Article Next-generation sequencing (NGS) has raised a growing interest in phage display research. Sequencing depth is a pivotal parameter for using NGS. In the current study, we made a side-by-side comparison of two NGS platforms with different sequencing depths, denoted as lower-throughput (LTP) and higher-throughput (HTP). The capacity of these platforms for characterization of the composition, quality, and diversity of the unselected Ph.D.(TM)-12 Phage Display Peptide Library was investigated. Our results indicated that HTP sequencing detects a considerably higher number of unique sequences compared to the LTP platform, thus covering a broader diversity of the library. We found a larger percentage of singletons, a smaller percentage of repeated sequences, and a greater percentage of distinct sequences in the LTP datasets. These parameters suggest a higher library quality, resulting in potentially misleading information when using LTP sequencing for such assessment. Our observations showed that HTP reveals a broader distribution of peptide frequencies, thus revealing increased heterogeneity of the library by the HTP approach and offering a comparatively higher capacity for distinguishing peptides from each other. Our analyses suggested that LTP and HTP datasets show discrepancies in their peptide composition and position-specific distribution of amino acids within the library. Taken together, these findings lead us to the conclusion that a higher sequencing depth can yield more in-depth insights into the composition of the library and provide a more complete picture of the quality and diversity of phage display peptide libraries. MDPI 2023-03-11 /pmc/articles/PMC10049078/ /pubmed/36982469 http://dx.doi.org/10.3390/ijms24065396 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Sloth, Ane Beth Bakhshinejad, Babak Stavnsbjerg, Camilla Rossing, Maria Kjaer, Andreas Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS |
title | Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS |
title_full | Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS |
title_fullStr | Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS |
title_full_unstemmed | Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS |
title_short | Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS |
title_sort | depth of sequencing plays a determining role in the characterization of phage display peptide libraries by ngs |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10049078/ https://www.ncbi.nlm.nih.gov/pubmed/36982469 http://dx.doi.org/10.3390/ijms24065396 |
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